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Almond characterization and bioanalysis for seed oil extraction

Characterization and bio analysis of almond seed
oil; a comparative study of conventional and advanced extraction techniques
Statement
of the problem
This study is designed for assessment of
conventional and advanced extraction methods used for extraction of almond oil.
This investigation is correspondingly motivated that in what way the progressive
methods cannot only reduced the employment efforts, time, space, cost and
complexity but also increase shelf life of extracted oil.
Objectives of this study
The aim/purpose of the study
were to select the extraction procedure which has cost effective, friendly
reaction with environmental conditions and yielded maximum oil having bioactive
compounds. The targets of the objectives of this research were based on the
following steps:
       
i.           
To introduce the various different expert extraction technique
for the oil extraction
     
ii.           
To enhance the extraction techniques in order to have
optimal conditions for optimization of maximum oil productivity
   
iii.           
To study the physicochemical standards in the direction
of assess the quality of oil
   
iv.           
Fatty acid profile of extracted oil to judge the effectiveness
of extraction technique
     
v.           
To evaluate the anti-oxidant and anti-microbial
activity of oil.
Significance of the
study
                      This study is very significant for market
demand in the fever of oil shortage for nutritional and medical purposes;
increase the productivity of almond oil using high throughput techniques and
methodologies. The multiple advantages are listed below

  •       i.   Less time consuming
  •      
    ii.   
    Low heating
  •    
    iii.   
    Reduce labor
  •    
    iv.     
    Commercially favorable


Outline of the
study
 This study comprises of following
                  i.          Synthesis of almond oil by advanced
(Microwave Assisted Exrraction and Ultrasonic Assisted Extraction) and
conventional (Soxhelt Assisted Extraction) techniques.
                        ii.           
Determination of physiochemical
parameters of almond oil.
                        iii.           Determination of antioxidant and
antimicrobial activity of almond oil.
                        iv.           
Characterization of defatted seed meal
of almond.
Introduction:  
History of lipid
In biochemistry,
lipids are small amphipathic, hydrophilic and hydrophobic biomolecules that
confirm the soluble mechanism of many non-polar solvents (McNaught & McNaught, 1997). The non-polar
functionalities are especially adapted to hydrocarbons (hydrogen + carbon),
which are used to dissolve many other natural molecules themselves, but they do
not readily dissolve in water with a wide range of molecules such as vitamins
A, D, E and K but soluble in fat. The nonpolar solvent also has a less complex
dissolution process with many natural compounds such as waxes, many fatty
acids, sterols, phospholipids, triglycerides, diglycerides and monoglycerides.
In an aqueous medium, certain lipids have amphiphilic properties such as
liposomes and multiple or unilemellar membranes of vesicles, so that the
biological concept of lipids consists wholly or partially of two heterogeneous
types of biochemical building blocks and subunits; isoprenic groups of ketoacyland (Fahy et al., 2005). Lipid self-synonym of fats,
which are subgroup known as triglycerides and consist of two major classes’
suifs and huiles respectively including with resins,
balsams, tallow , waxes, greases, oil and volatile oils with very inclusive
order (
Chevreul et al.,
1823
; McClements
et al., 2015
). Therefore the fats are simple lipid and made up of
glycerol and with fatty acids which are 
essential for flora and faunas due to its oily alimentary nature such as
carbohydrate (“saccharine”) and albuminous: alongside protein (Dugan et al 2011; Coley, 2004).
The class of lipid lipoid, lipin,
and lipid with different implications
such as
phospholipid including cephalen, sphingomyelin and cerebroside are found in
lecithins of hen egg and the human cerebrum
(
Spiller et al., 1998). In earlier study many other
important clusters of lipoids was etymologically originate from the Greek lipos
fat (
Bertrand, 1923),
and reported in three distinct categories like basic lipoids waxes and oils (genuine
waxes, sterols, alcohols), compounds and complex lipid with glycolipoids & phospholipoids
and derivative including with alcohols, sterols and fatty acids (
Christie & Han, 2010)
. The classification of lipids
comprehensive divides into diverse number of classes containing fatty acids,
sterol lipids,
polyketides,
saccharolipids, sphingolipids, glycerophospholipids and glycerolipids (
Fahy et al., 2005)
Classes
of lipid
Fatty acids 
Fatty acids and its
residues are very diverse group of chain-extension molecules made up of
hydrocarbon chains and end with
carboxylic acid group based on fatty acid synthesis
procedure as like acetyl co enzyme associate
with malonyl and methyl-CoA  (Brown, 2007). The
order of fatty acid conformation two ends
hydrophilic (polar) and hydrophobic (nonpolar) which are insoluble in water, if they have
double bound then geometric isoforms viewed based on chance of cis and trans
for conformation of molecules. The fatty acid class contain with structurally
complex molecules as building blocks such as carbon consist of saturated and
unsaturated form of chain with four and twenty four carbon in length and also
containing with halogens, oxygen, sulfur and nitrogen.
The illustration about biologically important fatty acids eicosanoids become
fundamentally significant in biological systems from eicosapentaenoic and
arachidonic acid with thromboxane, leukotriene’s, prostaglandins and
docosahexaenoic acid (
Holub,
2008
).
  





























































Picture1 (a):  LTB4 (an eicosanoid fatty acid,
illustration of leukotriene)


 I2 - Prostacyclin

Picture1 (b): I2 –
Prostacyclin
 (eicosanoid fatty acid,
illustration of a 
prostaglandin)

Glycero-lipids
The families of lipid
named glycerolipids are made up of three diverse substitute mono, di and tri
glycerols (Coleman & Lee 2004), and the
other name of glycerol fatty acid triesters
known as
triacylglycer and triglyceride in which three
groups are esterified via hydroxyl compounds. Therefore the lipid used to
stored energy in the form of fat in creature tissue and the hydrolysis of ester
of
triglycerides bond release glycerol for initial step
of metabolization of fat from adipose tissue (
Creighton,
1979
).
 triglyceride (C55H98O6)



Picture 2:  triglyceride
(
C55H98O6) of and unsaturated fat;
Left-hand: 
glycerol
and right handed: 
palmitic acidoleic acidalpha-linolenic acid
(from top to bottom)
Glycerophospho-lipids
The Glycerophospholipid
are present in nature commonly known as phospholipibds. The phospholipids show
intrinsic opportunity for cell and become key part of the cell lipid bilayer,
cell signaling and for metabolism of the cell (
Ali, 2014; Berridge & Irvine, 1989). They are also very vital for neural networks
and involved to change the arrangement of multiple heterogeneous
neurobiological disorders such as
involved function of three major components glycerophospholipid,
phosphatidylethanolamine and phosphatidylserine localized in biological
membranes (
Farooqui
et al., 2000
).
Phosphatidylethanolamine



                                                    Picture 3: Phosphatidylethanolamine 
Sphingolipids
The
Sphingolipids  are compound of very complex class that share many
structural feature for base backbone via sphingoid mutual understanding or
changed into glycosphingolipids,
ceramides, glycosphingolipids and many other compounds that formed
from de novo amino acid serine and a long-chain fatty acyl CoA. The essentially
sphingoid backbone of mammals are known as sphingosine and major lipid of mammals
known as 
sphingomyelins (Hori & Sugita, 1993)
Sphingomyelin

Figure 4: structure of Sphingomyelin
formed from de novo amino acid serine and a long-chain fatty acyl CoA
Sterol lipids
The sterol lipids combined with glycerophospholipids and sphingomyelins
are vital part of membrane lipids such as cholesterol and its derived compounds
show very diverse biological role rather than hormones and other signaling
molecules (
Bach & Wachtel,
2003
). The estrogen and androgens
families consist of eighteen and nineteen carbon respectively as like testosterone
and androsterone. On the other hand the progestogens class containing twenty
one carbons just as the glucocorticoids and mineralocorticoids (
Ireland & Wyatt, 1976). For example in the cleavage
process from secosteroids,  vitamin D is
important for central structure of B ring , bile acids and their conjugates (
Russell 2003), which in warm blooded animals are oxidized derivatives of
cholesterol and are produced in the liver.
Sterol



Picture5:
Sterol Chemical Structure
Prenol lipids
For the production of
phenol lipids
mevalonic acid pathways are important because they
produce five carbon originators as like isopentenylediphosphate and
dimethylallyldiphosphate (Kuzuyama & Seto, 2003).  The linear
alcohols and diphosphates are mainly produced via addition of five carbon units
and classified conferring to their teroene units, the higher number of carbon
containing like forty are known as polyterpenes. Carotenoid is an important
compound with basic isoprenoids and shows the ancestors of vitamin A function
as for antioxidants (Rao, 2007).



Picture
6:  2E- genraniol (Prenol lipid)
Saccharolipids
Saccharolipids are compound in which sugar
backbone is impotent for making well-marked structure with bilayer membrane. The
very familiar saccharolipids in 
Gram-negative
bacteria
are precursors
of lipid component of the 
lipopolysaccharides with acylated glucosamine (Pike,
2006
). With the help of conventional
enzyme polyketides are formed through using
acetyl and propionyl subunits polymerization, which share systematic
features with the fatty acid synthases.  
The saccharolipids have varied structural diversity with
natural products from fungal, marine sources, animal, plant and bacteria
involving large number of secondary metabolites
(Walsh, 2004; Caffrey
et al., 2008
).
Polyketides
The
polyketides molecules are exist in cycle nature but further its backbone can be
adjusted via several targeted compounds such as hydroxylation, oxidation,
methylation and glycosylation. The polyketide (polyketides) used for various
anti functional mediators between living things and many pathogen (microbes,
parasites and cancers) for example many companies launched internationally
products for anticancer, antimicrobial and antifungals named us doxycycline and
avermectins, tetracycline’s, erythromycins and antitumor epothilones (Minto & Blacklock, 2008).
Doxycycline



Figure7:
Doxycycline
For the process of fat production in
nature two main sources are available in the form of plant and animals, but for
animal lipid production is limited to fulfill their all requirement rather than
plant because plant rich with agricultural benefits and cultivated on large
scale in market so plants are main source of lipid production. The fats are
necessary for agricultural and food industries due to its daily based
utilization. For entire need of fats as for intrinsic or extrinsic activities
of humans, plant have a great significant on their origin and plantations in
everywhere of the world temperate and tropical regions. On the other contrast
animal have also great important as like one-sixth of the supply depend upon
human consumption in the form of meat and dairy products, but many seeds and fruits
like palm, olive, rapeseed, soybean and almond having oily nature are important
for lipid contents. Every plant oil has own mixture of fatty acids and other
component composition and available in market for greater efficiency of
anthropological consumption. 
Methods
for extraction of oil
The methods for
extraction depend upon nature of need and its gradients, but in which two
Chemical and physical methods are very important to obtaining oil from plant
seeds concerning via expression and extraction terminology, the term extraction
mean separation of liquid material from liquid solid mechanisms and expression
is a mechanical process in which liquid is extracted form liquid containing
solid (Khan & Hanna, 1983). The physical
and chemical both techniques have self-exploration for seed oil extraction
thereby chemical totally relies on solvent extraction (continuous solvent
extraction with machine pressing) and second physical method (bath hydraulic)
utilize mechanical power to extract oil from different plant seeds (Walkelyn & Wan., 2005).  Advance technologies in the field of oil
extraction form vegetable are more generalized as like supercritical-fluid
extraction (Pradhan et al., 2010).
                                                                                                             
Oil extraction “Cold pressed method”
The cold pressure is an
oil extraction technique depends on mechanical process in which mechanical
separation and expression methods are well-known. The earlier methods about oil
extraction was very hard and time consuming because oil extraction process was
followed by via hand using particular sponges and passed from any other
container, which are particularly absorb the oil from vegetables and  plants. Now a day’s in advance technology,
many modern methods for oil extraction are used in market such as using a
puncturing, spinning and centrifuge method to collect oil
from plant pulp and liquid materials of the plants.
Soxhelt Method
 The Soxhelt is oil extraction
method initially intended for extracting liquid from solid ingredients (
Soxhlet, 1879) in laboratory as its piece of apparatus (Harwood & Moody, 1989) this method have tree main mechanistic steps in the form of
particular sections such as percolator, thimble and siphon, this cycle can be
repeated many times over hour and days for better productivity in the form of
quantity and quality and each cycle have great versatile efficiency using
nonvolatile compound to liquefied in the solvent  (
Jensen, 2007).
Hydraulic press method
The hydraulic press
method is a manual method for oil extraction form plant seeds, where through
hand seed placed in equipment and distinguish the different layers of grains
using filter press paper or filter cloth and hydraulic force was applied via
cylinder. At the end clear the wastage of machine when oil extraction was
stopped and put the more fresh material for more productivity. This method was
very popular at the beginning of twentieth century for vegetables oil
extraction process but intensive use of labor was required, in later for
advancement of this method two main presses categories was introduced such as
open type (fresh material, required total of 30 minutes for batch working) and
closed type (oil seed unclosed by strong steel) (Williams,
2005
).
Mechanical pressing
The mechanical
pressing oil extraction process founded to be best technology for oil mining
and cake preparation via using helical screw in equipment.  The Mechanical pressing have a great
important for small farmer with low cost as well as free from toxic material as
compared to other earlier cited methods. This procedure founded to be much
optimized with optimum parameters as like moisture content of grains, auto
adjustment of temperature and minimum values of pressure applied for high
productivity (Pighinelli &
Gambetta, 2012
).
Solvent
Extraction
The solvent extraction
method is very efficient method for oil extraction via using solvent with low
content of seed such as soybeans and not applicable for other higher seed
contents like sunflower. This method also work very smoothly with optimum and
low temperature over screw pressing and give high quality of seed oil
production (Williams, 2005), but some
failure of this method are also attained such as solvent extract from certain
non-triglycerides and presence of volatile organic impurities (Michulec & Wardenki, 2005).
Supercritical-fluid (SCF)
extraction
In latter 1980 SCF
extraction method get popularity via showing advantage on oil assembly from
numerous of seed products such as cotton, sunflower, corn and soybean seeds via
using supercritical CO2. This method applied to take high productivity as like
hexane extractions with lighter color, low iron quantity and phospholipid
content (Temello & Guçlu-Ustundag, 2005).
In
final, the solvent extraction method is precise for commercial treatment based
on higher yield as like 97% with total control of technology but some failures
of this method are also accomplished such as solvent extraction from certain
non-triglycerides and presence of volatile organic impurities. The CSF method
applied to take high productivity as like hexane extractions with lighter
color, low iron quantity and phospholipid content. At the end the Mechanical
pressing method has a great important for small farmer with low cost as well as
free from toxic material as compared to other earlier cited methods. This
procedure initiated to be much augmented with ideal constraints as like
dampness content of grains, auto regulation of temperature and minutest values
of pressure applied for high productivity and avoid exploring the toxic results
on our atmospheres.
Refining
of oil
The
oil refinement is method in which edible oil must be refined before used in
which many undesirable ingredients are removed as like fatty acids and other fats
raw materials. The resultant product enhanced keeping properties such as clear
from smelly order, taste with suitable flour and color. The earlier alkali
methods for refinement of seed oil replaced through physical methods via
reducing chemical possession and introduced new steam refining method, in which
many additional ingredients are removed successfully such as pigment, heavy
metals, bleached and phospholipids. The quality of alkali and refined is match
closely with greater efficiency but oil refinement has greater advantage on
environment (
Cmolík &
Pokorny, 2000
). Furthermore the
different seed oil refinement methods (neutralization process, degumming
method, bleaching and deodorization process) are used to make comestible oil
for humans.
Degumming refining
The degumming oil
refining method follows the treatment mechanism with liquid H2O dilute acid
such as citric and phosphoric acid and show productivity via using centrifuge
in the form of gum continuing phosphate and then phosphate assemble as
rudimentary lecithin. The degumming method follows the various mechanistic
processes for seed oil refinement such as non-hydratable (NHP) and ethylene
diamine tetra-acetic acid
. After that the suitable lipases encourage
excruciating of the phospholipids into systems that are further voluntarily
separated and removed different harsh incidents (Gunstone., 2009).
Neutralization
The
neutralization is a process in which free fats (fatty acids) are removed via
using warm alkaline solution. The free fatty acids along with triglyceride
molecule react to oxygen and disturb the quality oil to convert rancid. However
this type of method during oil and soap solution neutralized the mixtures and
physically separates them, after that oil and soap are separated via washing to
remove traces of soap from oil (
Dahlke & Eichelsbacher, 1998).
Bleaching
The
bleaching is an non chemical impurities removal method in which mixture of oil
is prepared and removed unpleasant order and color with naturally occurring
bleaching clay without using any chemical reaction, subsequently to make clear
and clean oil.
Deodorization
The deodorization is an oil refining
process using vacuum steam distillation to removed unwanted taste and order. In
this method typically used high temperature to heat the oil for better quality (
Stage, 1981).
 literature  Study 
 In literature multiple
sequential
chemical and physical processes explored for oil distinction such as degumming,
neutralization, bleaching and deodorization. Further, the best establish and biologically
attractive method associated with neutralization for only palm oil is studied
with low level of phospholipid. This method has advantage on fewer loss of oil
and excessive yield for various other oils via shared with developed degumming processes
(Gunstone, 2009).
conventional oil extraction techniques
The conventional oil extraction is a very hard method in
time, space and complexity of environment, so the advance techniques are need
for oil extraction in the form of optimization. In early years the scientist
used the optimization process for oil extraction.

Status
of almond Oil in Pakistan

Pakistan is a developing country and its
growth rate of economy is 2.7%, so the growth requires high rate of energy but
country backbone is limited and original source of energy is plant seed oil. So
the Pakistan has oil reserves 0.31 billion barrels (bbl), however this amount
is not meet the demand of domestic usage because production of oil is only
59.08 thousand bbl per day and utilization is
426.72 thousand bbl per day
via based on 73%  import system
.
Pakistan is suffering in harden due to deficiency of oil, the reason of this is
inadequate power controlling mechanisms , fewer consideration actions in the
oil region, inappropriate supply of assets, corrupt ascendancy and poor
management of the rule and demand situation.
 
Pakistan can maintain the energy crisis via establish the technological
mechanistic process and techniques with the support of foreign investment and
well-designed government policies on energy, therefore Pakistan reconnoitering
novel sources, exclusively for almonds seeds to deal with oil crisis.
  


exportation of crude
Figure
9:
exportation of crude, refined and
seed oil

Almond and its
scientific detail (family and genera)

Almond
(prunus amygdalus) is a tree belongs to the rosaceae family and
its subfamily called prunoideae.
The total
production rate of family rosaceae is 246,345 and almond production is 26487. There are two main sort of the almond, one (Prunus amygdalus var. dulcis)
produced sweet almond and saccharine in taste for eating purpose (
Aremu et al 2010), but other
(Prunus amygdalus var. amara) is not sweet in taste or directly edible because
raw material after oil extraction produced various toxic acids like hydrogen cyanide
(
Herbst, 2007). The
genera (prunus genus) of almond tree are
categorized, which are including with many other plants sweet in taste such as
apricot, plums, cherries and peaches. The all mentioned plants hasve covered
seed with seed shell called hull, which contain edible seed inside the hard
shell of the plant seed as like almond. In northern of the temperature region
of lobe prunus (belonging rose family) are approximately separated in 430
species. The rosaceae (including rose family) is biggest flowering plant family
with 3400 species (Sathe at
al., 2002
). In botanic terms, almonds
is not a true nut and have hard stony shell covered on edible internal seed is
termed an endocarp (Cantor et al., 2006).
Almond seeds
Picture
10:
Almond seeds not a true nut and has hard stony shell covered
on edible internal seed
Globally Cultivation of Almond
Almond is very popular globally
distributed tree, in the area of United States and California the almond
products are widely available on industrial level and become largest almond
producer countries (
Maestri
et al., 2015
).  According to observation of food and
agricultural organization international productivity of almonds is between 1
and 1.5 million metric tons, the almond is also distributed in other word
largest market east and southern Asian zone such as Australia, Syria, turkey,
Pakistan, Portugal, Spain, Syria (
Maltsbarger, 2017; Rushforth, 1999). the production of sweet almond is mostly
produced from Valencia, Jordan, Spain, 
United States and California, but California have 70 crop rate and  great worth of exportation of almond with $
1.08 billion (
Kumar, 2018).
Production of almond in Pakistan
The almond in the region Baluchistan province of Pakistan is
largest almond producer market, during the middle of 2008 to 2009 Pakistan
cultivated almond on 11002 hectors and produced almost 26497 tons for
fulfilling the domestic demands. In Pakistan almond has great importance and
used for many propose such as in sweet dishes, vita oils, tibb unani therapies and
Indian Ayurveda.
Botanical Features
The almond is a tree belongs to
the rosaceae family and its subfamily called prunoideae. Almond
plants are very in sized small to medium, commonly 11 to 16 feet in commercial
orchards with 2 – 6 straightforward leaves (
Hortus, 1976). The almond flowers with five
petals and sepals are structurally very identical with numerous other related
tree families, they also sweet in taste such as peach and
Prunes flowers (Rushforth, 1999). In botanic
terms, almonds are not a true nut and have hard stony shell covered on edible
internal seed is termed an endocarp (Cantor et al.,
2006
).


The almond trees show self-incomplete pollination
process and for great productivity of blooming period almond also do cross
pollination with honey bee (
Huxley &
Griffiths 1999
). There are two
main sort of the almond; one produced sweet almond and saccharine in taste for
eating purpose, but other is not sweet in taste or directly edible because raw
material after oil extraction produced various toxic acids like hydrogen cyanide.
The genera of almond tree are categorized, which are including with many
other plants sweet in taste such as apricot, plums, cherries and peaches. The
all mentioned plants have covered seed with seed shell called hull, which
contain edible seed inside the hard shell of the plant seed as like almond.

Uses
of almond
         
The
almond has edible nuts and its product have a great importance in worldwide
commercial markets due to nutritional ingredients, therefore almond called
powerhouse of the nutrition for every type of living organism such as high
lipid content (45-65%) carbohydrates (15-20%) and proteins
(21%), vitamin A, B2 and E, calcium, fiber, folic acid, magnesium,

copper, niacin, potassium, iron and
zinc (Aremu et al., 2010; Herbst, 2007).
The Vitamin B1 (rich with mono saturated fat)
is also extracted from almond that is responsible for lowering cholesterol
level of humans. The almond has lipid content which is vital for protection of
structural component of cell membrane in the form of energy storage
(Fahy
et al., 2005; Subramaniam et al., 2011
),  and also very useful for nanotechnology, food
industries and cosmetic products. They also have significance in medicinal
field for cancer handling, increase effort of food through colon and recover
taint
(Davis & wahashi, 2001).
 In latest research, researcher show the evidence for almond
is a great impact on our routine diet such as usage of almond oil increase and
decrease the high and low level blood density via interaction of lipoproteins (Spiller et al., 1998). Almonds are very vital for
coverage of nerves system to treat the brain for intelligence level of human
and also enclose with triacylglycerols which as intracellular oil
precipitations in the cotyledon tissues of the nut (Ren
et al., 2001
).
almond seed test
Legends
SAE = Soxhelt Assisted Extraction                                 
MAE =Microwave Assisted Extraction                       
UAE =Ultrasonic Assisted Extraction
SEO =Soxhelt Assisted Extracted oil
MAEO =Microwave Assisted
Extracted oil                          

UAEO =Ultrasonic Assisted
Extracted oil

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